Bottom Line:
Although such ions offer tremendous analytical advantages, algorithms to decipher MS/MS spectra for the presence of diagnostic ions in an unbiased manner are currently lacking.To benchmark the software tool, we analyzed large higher-energy collisional activation dissociation datasets of samples containing phosphorylation, ubiquitylation, SUMOylation, formylation, and lysine acetylation.Using the developed software tool, we were able to identify known diagnostic ions by comparing histograms of modified and unmodified peptide spectra.

Mentions:
Having established that SPIID is capable of mapping composition-specific fragment ions such as immonium and diagnostic ions, we next wanted to investigate the algorithm's ability to perform more advanced spectral analyses. In recent years the analytical utility and diagnostic value of neutral losses induced by CID, electron transfer dissociation, or electron capture dissociation have been realized (51–53). A similar catalog of commonly observed neutral losses from HCD has also been described (54). However, to our knowledge no commonly available software tool has been described for the easy extraction of such neutral loss information. To demonstrate the ability of SPIID to extract such information, we first used high-resolution isotope spacing to deconvolute and deisotope each detected fragment ion to its singly charged counterpart for all HCD-generated MS/MS spectra. Next, all fragment ions were aligned to the parent mass (MH+) of the individual MS/MS spectra (Fig. 3A). All MS/MS spectra were then converted to negative m/z values that could easily, and in a single step, be interrogated for common neutral losses through spectral binning by SPIID (Fig. 3B). The results demonstrated that HCD generally did not induce many neutral losses relative to CID and electron capture dissociation/electron transfer dissociation, although a strong water loss and losses corresponding to intact amino acids were observed, with the latter associated with y-ion formation through regular backbone fragmentation. Next we investigated the same HCD-induced neutral losses in a phosphopeptide enriched sample by interrogating a sample derived from SCX and TiO2 enriched tryptic peptides (55, 56). Because of the chemical nature of the peptide phosphogroup, it easily detaches during collision-induced fragmentation, generating neutral losses corresponding to various types of phospo-groups, such as HPO3 and H3PO4. As expected, our SPIID analysis revealed prominent losses of phospho-groups, followed additionally by loss of H2O and combined losses (e.g. H2O + H3PO4 = H5PO5) (Fig. 3C).

Mentions:
Having established that SPIID is capable of mapping composition-specific fragment ions such as immonium and diagnostic ions, we next wanted to investigate the algorithm's ability to perform more advanced spectral analyses. In recent years the analytical utility and diagnostic value of neutral losses induced by CID, electron transfer dissociation, or electron capture dissociation have been realized (51–53). A similar catalog of commonly observed neutral losses from HCD has also been described (54). However, to our knowledge no commonly available software tool has been described for the easy extraction of such neutral loss information. To demonstrate the ability of SPIID to extract such information, we first used high-resolution isotope spacing to deconvolute and deisotope each detected fragment ion to its singly charged counterpart for all HCD-generated MS/MS spectra. Next, all fragment ions were aligned to the parent mass (MH+) of the individual MS/MS spectra (Fig. 3A). All MS/MS spectra were then converted to negative m/z values that could easily, and in a single step, be interrogated for common neutral losses through spectral binning by SPIID (Fig. 3B). The results demonstrated that HCD generally did not induce many neutral losses relative to CID and electron capture dissociation/electron transfer dissociation, although a strong water loss and losses corresponding to intact amino acids were observed, with the latter associated with y-ion formation through regular backbone fragmentation. Next we investigated the same HCD-induced neutral losses in a phosphopeptide enriched sample by interrogating a sample derived from SCX and TiO2 enriched tryptic peptides (55, 56). Because of the chemical nature of the peptide phosphogroup, it easily detaches during collision-induced fragmentation, generating neutral losses corresponding to various types of phospo-groups, such as HPO3 and H3PO4. As expected, our SPIID analysis revealed prominent losses of phospho-groups, followed additionally by loss of H2O and combined losses (e.g. H2O + H3PO4 = H5PO5) (Fig. 3C).

Bottom Line:
Although such ions offer tremendous analytical advantages, algorithms to decipher MS/MS spectra for the presence of diagnostic ions in an unbiased manner are currently lacking.To benchmark the software tool, we analyzed large higher-energy collisional activation dissociation datasets of samples containing phosphorylation, ubiquitylation, SUMOylation, formylation, and lysine acetylation.Using the developed software tool, we were able to identify known diagnostic ions by comparing histograms of modified and unmodified peptide spectra.